Method of treating hypertension in animals with an extract of barks of anacardiaceae

ABSTRACT

AN ANTI-HYPERTENSIVE FRACTION FOR TREATMENT OF ANIMALS IS EXTRACTED FROM FRESH POWDERED BARK OF ANACARDIUM OCCIDENTALE L (THE CASHEW TREE) BY MACERATION WITH WATER IN THE DARK AT 0-5*C.

METHOD OF TREATING HYPERTENSION IN ANIMALS WITH AN EXTRACT F BARKS 'OF ANACARDIACEAE Yvonne Thuillier, Paris, and Paulette Giono-Barher, born Michel, Crouy-sur-Ourcq, France, assignors to Albert Rolland S.A., Paris, France No Drawing. Continuation-impart of abandoned application Ser. No. 56,002, July 17, 1970. This application Sept. 29, 1972, Ser. No. 293,516

Claims priority, application Great Britain, July 19, 1969, p

36,427/ 69 Int. Cl. A611: 27/00, 27/14 US. Cl. 424-195 1 Claim ABSTRACT OF THE DISCLOSURE An anti-hypertensive fraction for treatment of animals is extracted from fresh powdered bark ofAnacardz'um occidentale L (the cashew tree) by maceration with'water in the dark at 0-5 C.

The presentinvention relates to a new extract prepared from the bark of the cashew-tree, Anacardium occidentale L, which is also known by the name of Anacardium senegalensis. The cashew-tree belongs tov the anacardiaceae family which also includes the sumac, th'e'pistachioj tree and the turpentine-tree.

Anacardium occidentale L or the"cashew tree.is a tree of North American origin, butlwidely planted inSenegal. The fruit, the anacard, cashew or cashew nut,.has a certain number of external medical uses, such as the treatthe mesocarp is allowed to run onto the leprous spots and,

antileprous ointments are applied to .the artificially produced sores produced by the juice. 1 v

The bark contains a gum which exudes if the tree is notched and which solidifies, changing from a red color to a yellow color. This gum is not used in the process of the invention. j v

It is known from the publication by Costra de Aguiar in the Brazilian review, Anais da Faculdade de Medecina da Universidade do Recife, volume 18, No. 2,,pp. 193'7 (195 8), that the bark of Anacardium occidentale L contains an agent producing hypoglycaemia'. According to this author, the hypoglycaemia-producing properties have been demonstrated in rats treated with an extract from 50 g. of bark in one litre of water, concentrated to 25%, and then diluted to A with an 85% strength solution of physiological serum, the final pH being 7. 1

We. have now surprisingly found, thatv the bark of Anacardium occidentale L possesses an antihypertensive agent that can be extrated by using a process different from that used to extract the hypoglycaemia producing agent. 1

The present invention provides a process'for the preparation of an antihypertensive extract wherein fresh powdered bark of Anacardium occidentaleL (Anacardium Senegalensz's), is macerated with water in the dark at a temperature of between-0 and 5 C. and the resulting aqueous extract separated from residual solids.

It is preferred to use distilled water in themaceration and to use a concentration of ground bark of between 20 and 50 g. per litre of distilled water. The aqueous extract is conveniently separated from residual solid by filtration and the aqueous extract then concentrated to give a dry extract which normally comprises 20-30% by weight of the original gum free powdered bark.

The bark, which has been finely powdered in a grindingsieving device may be suspended in distilled water at a temperature of between 15 and 25 C.,; with. stirring.

3,809,750 Patented May 7, 1974 Thereafter, the maceration is carried out with exclusion of light at a temperature between 0 and 5 C. After maceration, the suspension may be filtred whilst maintaining itat a low temperature, between 0 and 5 C., still with exclusion of light. The filtrate may be concentrated in vacuo and, after standing in darkness at a low temperature, lyophilized.

An extract obtained by dipping the bark in cold water and boiling for some time does not display any anti-hypertensive activity.

The invention relates to the treatment of hypertension,

in animals with the extract of the invention. The extract may be administered orally or by injection and the composition will then be liquid, e.g. comprising the extract in an aqueous solution.

The'examples which follow are given to illustrate the invention.

' EXAMPLE 1 100 g. of bark are ground using a sieving grid, to give 88 g. of a powdered product finer than sawdust. This is suspended in 4.4 l. of distilled water at a temperature of 20 C., giving a concentration of 20 g./l. The mixture is stirredfor about 5 minutes and maceration is then allowed to proceed in a cold cabinet at +2 C., with exclusion of light for 48 hours. The mixture is stirred every 2 to 4 hours during the day. After maceration, the mixture is filtered in the cold cabinet at +2 C., still excluding light, through a first filter disc (C5) to give a cloudy liquor and then through a less porous second filter disc EKS 2 to give -a limpid pink liquor.

' The filtrate, which has a volume of 3.8 liters, has a 'pH 5.8 and a solids content of 0.68% by weight, i.e. it

contains 25.64 g. total solids. The filtrate is concentrated in vacuo'ona waterbath at 25 C. until its volume is 380 ml., when the solids content is 6.8% by weight. After standing at +2 C., a precipitate forms. A homogeneous sample is filtered on a folded filter. The precipitate retained on the filter is converted into a brown gummy varnish'ori warming. The remainder of the solution, having a volume of 320ml, is lyophilized on plates. 20 g. of a bufi-colored product of very low density are obtained.

EXAMPLE 2 90 g. of bark are ground. During grinding, difiiculties arise, which may be due to the starting material still being too moist, which results in a slight tendency to carmelise on the grinding apparatus. In this case, ,two passes without the sieving grid are carried out. The shredded and ground product weighs 90 g. This is suspended in 4.5 l. of distilled water at 20 C., (a concentration of 20 g./l.), with manual stirring for about 5 minutes. Maceration is then allowed to proceed by standing the suspension in a cold cabinet at +2 C., with exclusion of light, for 9 days. The suspension is stirred twice daily; for a few minutes. After maceration it is filtered in a coldcabinet at +2 C., still excluding light, through a first filter disc to give a cloudy liquor and then through a, less porous second filter disc to give a limpid pink liquor. g

The filtrate, which has a volume of 4.2 1., has a pH of 5.25 and a solids content of 0.62% by weight. It is concentrated in vacuo on a waterbath at 25 C. until its volume is 400 ml., when it has solids content of about 6.5% by weight on standing at +2 C.; a precipitate forms. A homogeneous sample is filtered on a folded filter and the precipitate retained on the filter is converted'into a brown gummy varnish on warming. The remainder of the solution, having a volume of 320 ml., is lyophilized on'plates and 25 g. of a buff-colored product of very low density are obtained. The extract was studied by two-dimensional chromatography on cellulose powder with a 5 hour migration, using various solvents and dyestu'ifs to allow the active fraction to be detected.

This fraction is not a protein and contains very little amino acids.

Indole nuclei were detected by, p-dimethylaminobenzaldehyde in N hydrochloric acid solution. Violet-pink spots are observed.

The extract appears not to contain purine derivatives (violet-pink color with eosin), or reducing sugars on their phosphate esters (absence of an orange spot in the presence of picric acid). However, the color of the extract itself renders difiicult the determination of certain colors, especially that of the reducing sugars.

However, desoxyribosides, detected by a solution of cystein hydrochloride in 3 N sulphuric acid, very large amounts of thiols, disulphide, compounds containing phosphorus and finally of traces of aminoacids were detected.

No alkaloid reaction was found using the Draggendorf reagent.

The chromatograms of the extract of Anacardium occidentale L were compared with those of the renal extract described in British patent specification No. 991,491. The pink spot relating to the presence of indole nuclei of the present extract appears to correspond to the violet spot of the renal extract. The two extracts. also show similar spots confirming desoxyribosides. However, the renal extract contains reducing sugars, small amounts of disulphides and no thiols.

The extract according to the invention was subjected to pharmacological tests which demonstrated the antihypertensive properties.

The technique employed was that of Page-Patton- Ogden which involves brushing an exposed kidney with collodion, and removing the opposite kidney 1 to 4 weeks later. This technique theoretically produces chronic hypertensions in of the animals operated on.

Wistar rats weighing on average 230 g. at the time of the first operation are used in the test.

Blood pressure is measured in a Giono-Chevillard- Krauthamer oscillometer which allows the arterial pressure to be measured indirectly using the return flow of blood in the distal partof-an organ, in this case, the base of the tail of the rat,. :when thew-sleeve pressure is released.

The animal is kept'for' thirty minutes at a temperature of about 30 C. to produce a vasodilatation, the oscillometric sleeve being inposition from the start. It is necessary for the animal to" be absolutely immobile in order to carry out the measurements, and this sometimes requires a very long wait. An adjoining cage which allows several rats to be kept at 30 C. can be used and results in a considerable saving of time. v

The rats are anaesthetised with ether; after making an incision in the skin and the layer of muscle, the kidney is exposed and the conjunctive capsule is then carefully removed without damaging" the 'parenchyma. After carefully drying thekidney a -lay'er of collodion is. applied to the entire surface, the kidney, being kept suspended by a thread; several layers aiesuccessively applied, and the thread is then cut flush with the parenchyma. 10,000 units of penicillin are injected aim the operation and 8,000 units of penicillin on the following day; to days later, the nephrectomy of the untreated kidney is carried out.

A first series of tests was made'fto show that an extract of Anacardium-'occidentale according to the invention, administered to". normal, nonhypertensive rats, does not cause a substantial reduction in the arterial pressure.

A first group of control rats received, by injection, a physiological {saline solution; a second group of rats received, by injection, a physiological saline solution containing 50 g./liter of the; extract according to the invention, at a dose of 1 mg. per kg". of body weight. The arterial pressure (A.P.) wasmeasured by the oscillometric method [this measurement comprising the systolic hypertensive.

pressure (Tm)] and the diastolic pressure (TM)'before injection; then-4 to 5 .hours after injection. The results are given in Tables Ia and lb hereinafter, Table Ia giving the results of the measurements for the control rats, and Table lb the results of the measurements for the rats to which the extract according to the invention had been administered. p

A comparison of these tables shows that the arterial pressure, which was close to normal before the treatment, is not "modified by the injection of the Anacardium extract.

" 'Ase'condseries' of tests" was made to show the antih yperten'sive action of an Anacardium extract according to the invention, administered to rats made hypertensive by the Page" Patten and Ogden method described hereinabove (experimental hypertension).

The tests were made on three groups of experimentally hypertensive rats, to which a physiological saline solution containing 50 g./liter of the Anacardium extract according to the invention has been administered by injection, at a dose of 1 mg. per kg. body weight. The arterial. pressure was measured before injection and four tofive 'liours after injection. The results of these measurenie'nts are'g'iven for each group of rats in Tables IIa, IIb and He respectively. These tables show that the administration of the extract according to the invention provokes a marked reduction in the arterial pressure, ranging from -1ito"3 unitstfor the-systolic pressure (Tm) and 2 to 4 units for the distolic pressure (TM). This reduction in pressure, which is confirmed after 5 hours, is maintained for several daysafter injection.

The following tests are intended to demonstrate the difiere'nce existing between theantihypertensive Anacardium extractaccording to the invention and the hypo glycaemiant Anacardium extract obtained according to the method'de'scribed'by Costa de Aguiar (reference already mentioned). The extract obtained according to the inv'entionis passed over hide powder which is a reagent used by tanners for eliminating tannins; whilst the hypoglycaen'iiant .extract obtained according to de Aguiar retainsaall its ,hypoglycaemiant activity after passage over hide powder, the anti-hypertensive extract obtained according to the invention, when it has been free of the tannins by adsorption on hide powder, no longer presents any anti-hypertensiveactivity, as shown by the following tests.

A physiological" saline solution containing g. per litre of theAnac'ardium extract prepared according to "thein'yention' then "passed over hide powder, is injected atadose of 1 mg./kg. of body weight, to two groups of rats, one'no't hypertensive and the other experimentally The results of the measurement of the arterial pressure are given in Tables 111a and IIIb respectively for the hypertensive rats and for the non-hypertensive rats. These tables show:

Ontheonexhand, that the arterial pressure which was close to normal in the nonhypertensive rat is not modified .by injection of the product prepared according to the invention and then passed over hide powder; On the other, hand, that the product prepared according I to the invention and freed of the tannins by adsorption on hide powder has no antihypertensive activity. Thus, "the antihypertensive fraction is contained in the tannin fraction of the extract according to the invenvtion, whilst the hypoglycaemiant fraction of the extract accordingto -de Aguiar-is-not found in the tannins. W The. last ,series of tests shows the antihypertensive activity of: the product; prepared according to the invention, administered to. experim entally hypertensive rats, at the (same dose as, .the product modified by passage over hide powder in the preceding tests. Three groups of hypertensiverats received by injection, a physiological saline solution containing 100 g./liter of the product prepared ac- 1947 I. P. Lippinc'ott (30., Phila., Pa.

v sgsogyso 7 8 References Cited Husas Phafmaceutical' Dispensizi'gflth ed., pp. 630- 631, Mack Publishing Co.,Easton,-Pa. (1966). Anaxs da Faculdade de Medlcma da Umversdade do Rusby et aliMThe Properties and Uses of g f pp Recfe, vol. 18, No. 2, 193-7, 1958. ,4 v 6 l pp 21-7-2619, Blaklstons Son & Co. Inc., Phlla. Pa. 1930).

Martin et 211., Remingtons Practiceof Pharmacy, 11th ed., p. 246', TheMack Publishing Co; Eastcfi,Pa:"(195);, f;

Chemical Abstracts, vol. 10, p. 2786 (1916). a v. :5 Persinos et aL, Journal of Pharmaceqtical Sciences, AP T E Pnmary Exammer pp- 1 7)- 1 D, MOYER, Assistant Examiner The Dispensatory of U.S.= A. 24th ed.,-pp.-1181-1'183 V ,6 v

I us. C1.X.R.

- 1o The Merck Index,'"7th'ed.,1960,'pp.' 1010 and 1011. 524 480 Published by Merck & Co. 1nc., New Jersey. I

' "Dawes et aL, Brit. J. PharmaQcol. (1950), 5, pp. 65-76. 

